Hydrolysis process for the preparation of rubidomycin

ABSTRACT

The antibiotic rubidomycin, otherwise known as &#39;&#39;&#39;&#39;13,057 R.P.,&#39;&#39;&#39;&#39; is obtained by subjecting the antibiotic 13,213 R.P. to acid hydrolysis at a temperature between 10* and 75* C. for a length of time of from 48 hours to 30 minutes, the length of time varying inversely with the temperature. Rubidomycin and 13,213 R.P. are both described in British Pat. No. 985,598.

United States Patent Andre Belloc Hauts-de-Seine;

Yvan Charpentie, Paris; Jean Lunel, Paris; Jean PreudHomme, Paris, all of France [2]] App]. No. 711,495

[72] Inventors [22] Filed Mar. 8, 1968 [45] Patented Oct. 26, 1971 [73] Assignee Rhone-Poulenc S.A.

Paris, France [32] Priority Mar. 15, 1967 [33] France [54] HYDROLYSIS PROCESS FOR THE PREPARATION OF RUBIDOMYCIN 9 Claims, No Drawings [52] U.S. Cl 195/80, 424/123 [51] lnt.Cl Cl2d 9/00 [50] Field oi Search 195/80; 424/l l6,'120, 123

[56] References Cited OTHER REFERENCES Chemical Abstracts, Vol.60, 1964, p. 873b.

Primary Examiner.loseph M. Golian Attorney-Stevens, Davis, Miller & Mosher HYDROLYSIS PROCESS FOR THE PREPARATION OF RUBIDOMYCIN This invention relates to a process for converting the antibiotic designated by the number 13,213, R.P. into the antibiotic rubidomycin (otherwise designated 13,057 R.P.) by hydrolysis in an acid medium.

The specification of British Pat. No. 985,598 describes the antibiotic designated by the number 9,865 R.P., its three principal constituents designated by the numbers 13,213 R.P., 13,057 R.P. and 13,330 R.P., the aglycone of 9,865 R.P. designated by the number 13,567 R.P., the preparation of the antibiotic 9,865 R.P. by cultivating aerobically Streptomyces 8899" (NRRL 3046) or Streptomyces 31,723 (NRRL 3045) in an appropriate nutrient medium, and the separation of antibiotic 9,865 R.P. into its constituents, as well as the preparation of the aglycone of 9,865 R.P. by hydrolysis of 9,865 R.P. or of rubidomycin.

The antibiotic 9,865 R.P. on average contains 30 percent by weight of rubidomycin and 30 to 40 percent by weight of 13,213 R.P., but these percentages may vary within wide limits depending on the conditions under which 9,865 R.P. is prepared.

The two principal constituents of 9,865 R.P., i.e. 13,213 R.P. and rubidomycin, are of particular interest because of their very pronounced anti-cancer activity. 13,213 R.P., whilst being about 100 times as active as rubidomycin in animals, is also 100 times more toxic; furthermore 13,213 R.P. suffers from the disadvantage of not being crystalline whereas rubidomycin is easily crystallisable. It is therefore desirable to remove 13,213 R.P. from rubidomycin utilized in vivo.

The conversion of 13,213 R.P. to rubidomycin thus has the advantage of increasing the production of rubidomycin and of removing an undesirable substance from the finished produce suitable for therapeutic application.

The two constituents of 9,865 R.Pr (13,213 R.P. and rubidomycin) contain the same chromophoric grouping or aglycone, designated by the number 13,567 R.P., and their constitution may be schematically represented as follows:

13,567 R.P. aminated sugar rubidomycin rubidomycin +sugar 13,213 R.P.

Hydrolysis in an acid medium under appropriate conditions causes the following two reactions:

13,213 R.P. rubidomycin rubidomycin 13,567 R.P. aminated sugar.

It is thus necessary to determine the conditions of transformation which make it possible to obtain the maximum amount of rubidomycin and the minimum amount of 13,567 R.P.

According to the present invention, a process for the preparation of rubidomycin comprises hydrolysing a solution of 13,213 R.P. with an acid in aqueous solution at a temperature between and 75 C. for a length of time of from 48 hours to 30 minutes, the length of time varying inversely with the temperature, and separating rubidomycin from the reaction mixture by any of the usual methods for isolating and purifying antibiotics. The hydrolysis reaction is preferably carried out for a period of from 1 hour to hours at a temperature of from C. to 50 C., the length of time varying inversely with the temperature.

The 13,213 R.P. may be in aqueous solution or, as is preferred, in an organic or aqueous-organic solvent medium, or in a fermentation medium wherein the antibiotic has been formed by cultivating Streptomyces 8,899 or Streptomyces 31,723. Organic solvents which generally may be used are alcohols containing 1 to 4 carbon atoms such as methanol or butanol, ethers such as dioxan or tetrahydrofuran, ketones such as acetone or methyl ethyl ketone, esters such as ethyl acetate, chlorinated hydrocarbons such as methylene chloride or chloroform, or a mixture of these solvents.

The hydrolysis of 13,213 R.P. is efiected with inorganic acids (e.g. hydrochloric, phosphoric and sulfuric acids) or organic acids (e.g. acetic and oxalic acids) at various concentrations, but hydrochloric acid or oxalic acid are preferably used at normalities offrom 0.01N to IN.

The concentration in the reaction medium of 13,213 R.P. to be converted to rubidomycin may vary from 0.01 percent 5 percent by weight.

It is necessary to be able to determine rubidomycin, 13,213 R.P. and 13,567 R.P. in aqueous, organic or aqueous-organic solutions, in the crude products, in the semipurified products and in the pure products in order to determinethe optimum hydrolysis conditions and the composition of the final products.

The techniques for their determination are based on the differences in the physico-chemical and biological properties of the different constituents.

Partition chromatography on cellulose or absorption chromatography on silica gel in various solvent systems, such as mixtures of alcohols of low molecular weight and water, or mixtures of alcohols of low molecular weight, esters of aliphatic acids and water, may be used as rubidomycin, 13,213 R.P. and 13,567 R.P. have different Rf values.

As rubidomycin and 13,213 R.P. are naturally colored, it is possible to separate them by thin layer chromatography or by chromatography on a column, and either to determine them spectrographically in relation to a standard or, more simply to convert the separated products to the aglycone 13,567 R.P. by severe hydrolysis, to determine the percentage of the chromophoric group and hence to deduce the content of each of the constituents.

Furthermore, the biological activity in vitro of rubidomycin and 13,213 R.P. is different, and the aglycone is inactive. If, by definition, pure rubidomycin in the form of the base corresponds to 1,000 g/mg. then pure 13,213 R.P. corresponds to 60,000 g/mg. when determined turbidimetrically, using Klebsiella pneumoniae as the sensitive micro-organism. This difference makes it possible not only to carry out determinations on the crude products and on the solutions but also to detect very small quantities of 13,213 R.P. in purified batches of rubidomycin. In order to carry out these detenninations, it is sufficient to chromatograph the products as indicated above and to develop the chromatograms by bioautography on a nutrient agar plate inoculated with Bacillus subtilis or Klebsiella pneumoniae.

The following Examples, in which the indicated temperatures are in degrees Centrigrade, illustrate the invention. The yields given in the Examples are defined as follows:

Yie1d=We1ght of rubidomycin contained in the product obtained Weight of rubidomycin contained in the treated pr0ductX100 EXAMPLE 1 Two solutions containing 5 mg./cc. of pure rubidomycin and of pure 13,213 R.P. are prepared in an ethyl acetatemethanol mixture (1:1 by volume). An amount of N- hydrochloric acid corresponding to 4 cc. per gram of antibiotic employed is added to each solution. The solutions are heated to 25 and that temperature is maintained with agitation. At certain intervals of time (1 hr., 4 hrs., 9 hrs. and 20 hrs.) 0.5 cc. of each of these solutions is withdrawn, and a precipitate formed by addition of diethyl ether (10 cc.). The resulting precipitates are filtered off, drained and dried. Their strength is measured by circular chromatography on Arches No. 302 paper impregnated with a phosphate buffer at pH 4.8 using n-butanol saturated with water as the development solvent. The visible zones corresponding to rubidomycin, 13,213

Percent yield determifned asatunction EXAMPLE {V Constituents of or? Eight fractions of l g. of crude antibiotic 9,865 R.P., con- Product determined hr. 1 hr. 4 hrs. 9 hrs. 20 hrs. mi i 345 pig/mg f bid i d 400 Lg/mg f Rubidomycm {Rubidomycin 100 90 go 90 33 13,213 R.P., are dissolved in eight quantities of 5 cc. of Egg g Lg kg g chloroform. To each fraction added an n-butanol-6N 13,213 1 1 };i: 5 i 31 37 55 hydrochloric acid mixture (96:4 by volume; 10 cc.). These 13,567 0 2 3 4 10 solutions are heated to or to and maintained at that temperature with agitation. The composition of one fraction EXAMPLE n 10 kept at 20. and of one fraction kept at 30 is determined at regular intervals of time (2, 3, 8, and 16 hours). For this pur- Two solutions containing 3.25 mgJcc. of pure rubidomycin pose, an o -he ne mixture (1:1 by volume; 150 cc.) is and of P 13,213 are p p in a S01v6!l1 mixture of added to each fraction in order to precipitate the rubidomycin hyl eta -m han l 11 by v m An m n f N and 13,213 R1. The resulting products are filtered off, hydrochloric acid corresponding to 4 cc. per gram of pro 15 drained, dried and determined colorimetrically in order to emp oye i added 1 each Solution. Th i fi 111 11 evaluate the percentage of rubidomycin, and turbidimetrically are heated to 37 and that temperature maintained with agitawith Klebsiella pneumoniae as the sensitive micro-organism in tion. 30 Microlitres of each of the solutions are withdrawn at order to determine the content of 13,213 RP. regular intervals of time (1 hr., 2 hrs., 4 hrs., 6 hrs., and 24 The following table summarizes the results obtained:

Characteristics of the products obtained Ruhidomycin 13,213 R.P. Yield Percent Temperature Duration Weight, Strength, Weight. Strength, Weight, rubid- Percent C. in hours g. ig/mg. g. pig/mg. g. omycin 13,213 R1. 0 1 345 0. 345 400 o. 400 100 100 20 4 0. 78 630 0. 400 90 0. 063 141! I6 8 0. 78 670 0. 520 68 0. 053 150 13 16 0. 73 710 0. 515 60 0. 042 1411 10 0 1 345 0. 345 400 0, 400 100 100 2 0. 7 665 0. 465 76 0. 053 135 13 30. 4 0.7 670 0.469 55 0.038 136 [1,5 8 0. 72 670 0. 480 41 0. 0291 130 T 16 0. n 745 0. 445 33 0. 020 1211 6 hrs.) and deposited on a thin layer of silica gel impregnated 35 with a phosphate buffer at pH 7 The chromatograms are developed with the solvent phase of a mixture of n-butanolethyl acetate-water (5:5:2.5 volume). The zones of silica gel EXAMPLE v which contain the separated constituents are collected by scraping. These fractions of silica gel are eluted with a A product (46 g.) containing 150 pglmg. ol rubidomycin,

methanol-hydrochloric acid mixture (9:1 by volume). The eluates are heated at 50 for 1 hour 30 minutes in order to hydrolyse the two constituents, namely rubidomycin and 13,213 R.P., to give 13,567 R.P. The absorption ofthese solutions is measured at 480 nm and the contents of rubidomycin, 13,213 R.P. and 13,567 R.P. deduced therefrom.

The following table summarizes the results obtained:

650 rig/mg. of 13,213 RF. and 200 gig/mg. of 13,567 R.P. (a residual product from the fractionation of 9,685 R.P. of which the greater part of the rubidomycin has been removed) is dissolved in a methanol-ethyl acetate mixture (1:1 by volume;

Percent yield determined as a function of time Two solutions containing 5 mgJcc. of pure rubidomycin and of pure 13,213 R.P., are prepared in a methanol-ethyl acetate mixture (1:1 by volume). Amounts of N hydrochloric acid corresponding to 2 cc. and 8 cc. per gram of product employed respectively are added to aliquot fractions of these solutions. These solutions are treated as indicated in example 11 and the following table summarizes the results obtained.

1,840 cc.). N hydrochloric acid (55 cc.) is added, and the temperature raised to 37 and maintained there, with agitation, for 10 hours. The solution is concentrated at 30 under reduced pressure (20 mm.Hg.) to 500 cc. Butanol (500 cc.) is added and the solution again concentrated to 500 cc. Addition of hexane (5,000 cc.) to the butanol solution so obtained causes a precipitate to form. The precipitate is filtered off, washed, drained and dried. A product (42 g.) is obtained, of

Percent yield determflned as a function of time Constituents N HCl, cc./g Product treated determined 0 hr. 1 hr. 2 hrs. 4 hrs. 6 hrs. 24 hrs.

- Rubidomycin 100 100 97 79 55 "113,561 R.P 0 0 s 21 is 2 13,213 R.P 98 78 67 36 20 0 13,213 11.1 Rubidomy cu 1 2O 30 5 1 66 lflgjgidR. 1 2 3 1O 15 u i omycii 100 1 8 95 75 ""'i13,567 RR 0 2 5 15 25 5s 8 13,213 R.P 98 36 24 18 13,213 R.P Rubidomy 1 63 70 63 40 38 13,567 R .P 1 1 6 19 35 34 Properties Rubidomycin 13,213 R.P. 13,567 12.1% Weight, Strength, Strength, Strength,

Products g. gJmg. Weight, g. pgJIng. Weight, g. gJmg. Weight, g.

Untreated 46 150 6.9 650 21). 2 200 9. 2

Obtained after treatment. 12 440 18. 5 310 13 250 10.5

absorption maximum at 288 m l-E, cm. =150 absorption maximum at 472 m,u.-E cm. =20) EXAMPLE VI absorption maximum at 487 my.-E cm. =209 The product obtained in example V (10 g.) is dissolved in absorption maximum at 523 ml" E1l% cm'=l 16 the system of n-butanol-M/l 5 phosphate buffer at pH 6.5 (200 cc.). A counter-current distribution entailing 50 transfers is EXAMPLE vm carried out in a 50 cell Craig apparatus. Three fractions (cells A tibiotic 9,865 R.P. (463 g.) in the form of the crude base 0 to 14, 14 to 40 and 40 to 50 are made. The three fractions 20 taini g 330 gjmg. of rubidomycin and 330 ug/mg. of are treated identically. The aqueous phases are decanted and 13,213 R P i di l d i hl f 23 1 A "butanolthe butanol phases are diluted with hexane(l volume) and ex- 6 N h d hloric acid mixture (96:4 by volume; 4.6 l.) is tractcd three times with A f th V l of Watefl The q added with stirring, and the solution is heated at 30 for 15 Ou ex r c are Combined and eXlraCled twice at P" with hours. A first crop of antibiotic hydrochloride is precipitated 76 ofthe volume of chloroform. The mixture is washed at pH 8 25 b ddi i of acetone (35 1.) Thi precipitate i fil d ff, with 1/10 of the volume of water. It is then concentrated to wa h d and dried, A econd ro is obtain d by addition of 100 cc. and a precipitate formed by the addition of h X hexane (42.1.) to the mother liquors of the first crop. The The precipitates are filtered Washed and precipitate is filtered off, washed and dried as in the case of dried. the first crop.

The following Table summarizes the results obtained:

Rubldomycin 13,213 R1.

.. Percent Weight, Strength, Weight, Strength, Weight, I yield of Products g. lg/mg. g. gJmg. g. rubidomycin Untreated 463 330 153 330 153 Obtained: I

1st crop 130 860 15s 15 1.5 10.1} 5 2nd crop 58 532 32 1O 1). 5 19. 5

The results obtained are summarized in the following table:

Rubldomycin 13,213 Ry. 13,567 R.P.

Weight, Strength, Weight, Strength, Weight, Strength, Weight. Products g. gJmg. g. rig/mg. g. g/mg. g.

0 4.4 310 3.1 250 2.5 Untratem 1% 5 10 1. as so 0. 09 30 0. 045 obtflmed- 1. 1 325 0.91 150 0. 1s 25 0. e275 5. 2 100 0. 52 355 1. 75 545 2. 84

EXAMPLE V1] The product obtained in example V1 (0.5 g.), containing 910 ugJmg. of rubidomycin, is dissolved in a dioxan-water 55 EXAMPLE [X mixture (8:2 by volume; 5 ml.). The pH is adjusted to 4 by addition of concentrated hydrochloric acid (0.1 cc. Dioxan cc.) is added slowly with continuous stirring, and rubidomycin hydrochloride crystallizes. The precipitate is filtered off, washed with dioxan (5 cc.) and dried at under reduced pressure (0.5 mm.Hg). Rubidomycin hydrochloride (0.480 g.) is obtained; it has the following properties:

biological strength =95 5 ug/mg. elementary analysis: C=55.35%, H=6.45%, 0 =30.1%,

N=2.55%, Cl =6.05%.. ultraviolet spectrum: (determined on a solution in 96 percent ethanol) absorption maximum at 236 my-E, cm. =645 absorption maximum at 250 my.E, cm. =442 The hydrochloride obtained as described in example V111 (187 g.), containing 860 ugJmg. of rubidomycin and 15 ug./mg. of 13,123 R.P., is dissolved in methanol (360 cc.). Water (57 cc.) and dioxan (1.38 l.) are added. The mixture is clarified by decantation and slowly poured into dioxan (12.6 1.) with stirring, thereby causing crystallization. The crystalline hydrochloride product is filtered off, drained. washed and dried. A second crop is obtained by concentrating the mother liquors to 2 litres and adding hexane (2 1.). The product is isolated after filtering, washing and drying.

The following table summarizes the results obtained:

The rubidomycin hydrochloride thus obtained is identical to that obtained in example V11.

EXAMPLE X 5 A broth (200 1.) is divided into two identical fractions A Rubidomyt'in 13,213 R.P. Yield, percent Volume strength, Strength, Various (1.) or gJml. or Weight, ,ugjml. 01' Weight, Rubido- 13,213 phases weight (g) gimg. g. gjmg. g. mycin R.P.

BrothAorB 100 43.8 138 30 3. Fi1trateA 123 47.2 5.8 6.11 0.85 132 28 B taken up again 1 165 26 4.3 17. 5 2.51 18 16 Product A 1 2 5, 45 S75 4. 78 1211 0. 65 110 21. 5 Product B r 2 7 510 3. 57 325 .2. 28 81. 5 76 1 Litres. 2 Grams.

2 w l t and B, containing 43.8 ugjml. of rubidomycm and 30 ,ug/ml. e c mm of 13,213 R.P. Fraction A is subjected to an acid hydrolysis and fraction B, which is not subjected to an acid treatment, serves as a comparison standard.

Oxalic acid (30 g./1.) is added to fraction A. This mixture is 25 heated to 50, and maintained at that temperature for 1 hour, and then filtered in the presence of a filtration aid. The product is washed with water at 50 and the filtrate thus obtained (123 1.) is cooled to =5. The pH is adjusted to 4.5.

The broth fraction B is filtered in the presence of a filtration 30 ture (3:2 by volume; 50:1.) containing oxalic acid (1 kg.). The 35 mixture is stirred for half an hour, and filtered. Washing is carried out with a methanol-water mixture (1:1 by volume; 20 1.). The combined filtrates and washings (165 1.) thus obtained are cooled to +5 and neutralized to pH 4.5.

The filtrates A and B are thereafter separately treated as follows:

Each filtrate is passed through a column containing Amberlite RC 50 1.) in the acid form. The filtrate passes upwards through the Amberlite bed at a rate of 15 litres/hour. The column is thereafter washed with water (50 l.) in the case of filtrate A and with 50 percent methanol (50 l.) in the case of filtrate B, using 15 litres/hour circulating upwards, and then with a solution of methanol containing percent of water (75 1.) circulating downwards at a rate of litres/hour.

The washings are discarded, and the column is then eluted with a solution of the following composition: sodium chloride (10 g.), water (100 cc.) and methanol sufficient to make up 1,000 cc. The eluate, which contains the greater part of the antibiotic, has a volume of 40 litres. 1t is concentrated to a volume of 5 litres under reduced pressure mmllg.) at

1. Process for converting the antibiotic 13,213 R.P. into rubidomycin which comprises treating a solution containing 13,213 RP. with an acid in aqueous solution for between 48 hours at 10 C. and 30 minutes at 75 C. to hydrolyse 13,213 R.P. to rubidomycin, and separating rubidomycin from the solution.

2. Process according to claim 1 in which the solution of 13,213 R.P. is treated with an acid selected from hydrochloric, phosphoric, sulfuric acetic and oxalic acids in aqueous solution.

3. Process according to claim 1 in which the solution of 13,213 R.P. is treated with an acid selected from hydrochloric and oxalic acids in aqueous solution.

4. Process according to claim 3 in which the solution of 13,213 RF. is treated with a 0.01N to 1N aqueous solution of the acid.

5. Process according to claim 1 in which treatment of the solution of 13,213 R.P. with an acid in aqueous solution is carried out for between 15 hours at 20 C. and 1 hour at 50 C.

6. Process according to claim 1 in which 13,213 R.P. is in solution in at least one organic solvent selected from alcohols containing 1 to 4 carbon atoms ethers, ketones, esters and chlorinated hydrocarbons.

7. Process according to claim 1 in which 13,213 R.P. is in solution in at least one organic solvent selected from methanol, butanol, dioxan, tetrahydrofuran, acetone, methyl ethyl ketone, ethyl acetate, methylene chloride and chloroform.

8. Process according to claim 1 in which 13,213 R.P. is in solution in a fermentation medium wherein the antibiotic has been formed by cultivating Streptomyces 8,899 (NRRL 3,046) or Streptomyces 31,723 (NRRL 3,045).

9. Process according to claim 1 in which the solution of 13,213 R.P. treated with the aqueous acid contains from 0.01

percent to 5 percent by weight of 13,213 R.P. 

2. Process according to claim 1 in which the solution of 13,213 R.P. is treated with an acid selected from hydrochloric, phosphoric, sulfuric, acetic and oxalic acids in aqueous solution.
 3. Process according to claim 1 in which the solution of 13,213 R.P. is treated with an acid selected from hydrochloric and oxalic acids in aqueous solution.
 4. Process according to claim 3 in which the solution of 13,213 R.P. is treated with a 0.01N to 1N aqueous solution of the acid.
 5. Process according to claim 1 in which treatment of the solution of 13,213 R.P. with an acid in aqueous solution is carried out for between 15 hours at 20* C. and 1 hour at 50* C.
 6. Process according to claim 1 in which 13,213 R.P. is in solution in at least one organic solvent selected from alcohols containing 1 to 4 carbon atoms, ethers, ketones, esters and chlorinated hydrocarbons.
 7. Process according to claim 1 in which 13,213 R.P. is in solution in at least one organic solvent selected from methanol, butanol, dioxan, tetrahydrofuran, acetone, methyl ethyl ketone, ethyl acetate, methylene chloride and chloroform.
 8. Process according to claim 1 in which 13,213 R.P. is in solution in a fermentation medium wherein the antibiotic has been formed by cultivating Streptomyces 8,899 (NRRL 3,046) or Streptomyces 31,723 (NRRL 3,045).
 9. Process according to claim 1 in which the solution of 13,213 R.P. treated with the aqueous acid contains from 0.01 percent to 5 percent by weight of 13,213 R.P. 